This proposal addresses the mechanism by which the MCM2-7 helicase unwinds DNA at eukaryotic replication forks, a crucial, unsolved problem in the field of chromosome duplication. Prokaryotic replicative DNA helicases generally function as a single hexamer that translocates along one strand and excludes the other strand. However, the MCM2-7 complex may not fit this simple paradigm. In the G1 phase of the cell cycle, MCM2-7 is recruited into pre-Replication Complexes (pre-RCs) as numerous inactive dimers that encircle double-stranded DNA. At the G1/S transition, two protein kinases (CDK and DDK) cooperate with a large number of additional factors to activate the MCM2-7 helicase in a reaction whose mechanism is currently mysterious. Only a subset of MCM2-7 complexes is normally activated, raising the question of what happens to the other MCMs that are clamped tightly around dsDNA. To study the properties of the MCM2-7 complex in the context of vertebrate DNA replication, the applicant's laboratory employs a soluble cell-free system derived from Xenopus laevis eggs. A unique feature of this system is that it supports efficient replication of model DNA templates such as plasmids and;phage DNA, both of which can be readily modified in specific ways. We will combine this cell-free system with DNA nano-manipulation and real-time single molecule imaging to address the following questions. First, using stretched, doubly- tethered lambda DNA as a substrate, we will determine how many MCM2-7 complexes are present in each pre-RC and whether unactivated MCMs are removed or mobilized when struck by a moving DNA replication fork. Second, we will collide the replisome with strand-specific DNA roadblocks to address whether MCM2-7 translocates along ssDNA or dsDNA during DNA unwinding. Finally, we will use single-molecule imaging approaches to probe the dynamic properties of the MCM2-7 helicase complex. Of particular interest is whether the rate of MCM2-7-mediated DNA unwinding slows down when the helicase uncouples from the replisome during replication stress. Together, our experiments will elucidate the molecular mechanism and dynamics of the MCM2-7 helicase and thereby greatly deepen our knowledge of how cells copy and maintain their genomes. ) PUBLIC HEALTH RELEVANCE: Narrative To maintain the integrity of our genomes, it is crucial that cells make one precise copy of their DNA before each cell division ("DNA replication"). One of the most important enzymes involved in DNA replication is a "helicase" that separates or unwinds the two strands of the DNA double helix so that each strand can be copied. Although the helicase in higher organisms has been identified, we don't understand how it unwinds DNA. In this proposal, we use a series of innovative biochemical experiments to address this question. The work is relevant for human health because it appears that defects in DNA unwinding can cause genome instability and cancer.